Effect of the β-tricalcium phosphate particle on dental enamel associated with 10% carbamide peroxide bleaching

Background Since bleaching gels can cause adverse effects on tooth enamel, it is important to evaluate new remineralizing agents on the market and their effects. Material and Methods Seventy-five bovine enamel/dentin blocks (4x4x3mm) were randomly divided into six groups (n=10): Negative Control (NC) with no bleaching treatment or brushing; 10 CP (Carbamide Peroxide) (no brushing - Whiteness Perfect FGM); CT12 + 10 CP (Colgate Total® 12); ES + 10 CP (Elmex® Sensitive); BPC + 10 CP (Bianco® ProClinical); CMP + 10 CP (Colgate® Máxima Proteção Anticáries). The color was evaluated by reflectance spectrophotometry (∆E*ab, ∆E00, and ∆WID) at times T1 (baseline), T2 (24 hours after brushing), and T3 (24 hours after bleaching). Knoop microhardness (KHN) analysis were performed at T3. The enamel surface was qualitatively analyzed by Scanning Electron Microscopy (SEM). The data were analyzed using generalized linear models through descriptive and exploratory analyses, and a significance level of 5% was considered. Results Significant differences were observed when the bleached groups were compared to the NC group for ∆E*ab, ∆E00, and ∆WID at time T3 (p= <0.0001). However, the bleached groups presented no significant differences regarding studied times (p> 0.05). KHN did not differ significantly among the six groups (p=0.7585). Conclusions Toothpastes with tricalcium phosphate (β-TCP) do not intervene with the efficacy of bleaching treatment with 10% carbamide peroxide. Although a slight mineral deposition on enamel surface can be observed on SEM images, KHN was not significantly altered, and the polishing of the samples were maintained. Key words:Dental Bleaching, Carbamide Peroxide, Hydrogen Peroxide, Dental Enamel, Tricalcium Phosphate.


Introduction
At-home bleaching using low-concentration gels, preferably 10% carbamide peroxide (10 CP), is a highly recommended bleaching technique due to its easy application and reduced chair time and costs (1,2).While these gels have CP as an active component, hydrogen peroxide (HP) is the active factor responsible for teeth whitening (3).Their whitening mechanism comprises the dissociation of the 10 CP into HP, corresponds to one-third of the CP percentage (3,35% HP), and urea (6,65%) (1).Due to its low molecular weight, HP penetrates mineralized tooth tissues by diffusion (4).Then, it dissociates into water and oxygen radicals, which react with organic chromogens at dentin (5), promoting their breakdown by a redox reaction (6).The breakdown of the organic chromogens decreases light absorption and allows them to be removed from the tooth structure, resulting in whiter and less yellow teeth (7).However, many studies have shown adverse effects when using these oxidative agents, such as calcium and phosphate mineral loss (8) resulting in changes in the enamel's physical and topographical properties (9).These deleterious effects are caused by HP (8) and can be reversed through the application of remineralizing agents, in charge of calcium and phosphate ions deposition on the enamel surface (10)(11)(12).In this context, toothpastes have been improved to help minimize problems affecting the oral mucosa and dental elements (13).Therefore, dental treatments are being performed in association with several active ingredients incorporated in different toothpastes that have possible remineralizing potential in the face of erosive challenges (14,15).Tricalcium phosphate (β-TCP) is an active factor that has recently been introduced (16).It acts as a remineralizing source (17) since it dissociates into Ca 2+  and PO 4 3+ ions, essential for dental remineralization, making them available in the environment as single ions or their agglomerates (18).In addition, this active ingredient has reduced solubility when compared to other calcium and mineral salts, allowing it to act as a source of mineral components (19).Due to the modern population's lifestyle, more acidic diet, and constant search for a more harmonious aesthetic (20)(21)(22), studies on remineralizing agents are fundamental (23).Consequently, it is important to evaluate the remineralizing potential of toothpastes containing the β-TCP particles in the context of supervised home bleaching with 10 CP, since there are no in vitro studies on their effects on bleaching techniques with low-concentration gels.Therefore, this study aimed to evaluate the effect of two commercial toothpastes containing β-TCP in their formulation, when applied before bleaching with 10 CP, and the capability of promoting changes in dental enamel color and microhardness, to determine whether it contributes to minimizing the deleterious effects of low-concentration bleaching gels based on 10 CP.The null hypotheses tested were: (1) The evaluated commercial toothpastes would not change the teeth color after brushing followed by whitening with 10 CP; (2) Toothpastes containing β-TCP would not change the microhardness of dental enamel after brushing followed by bleaching with 10 CP; (3) The dental enamel's superficial topography would not be altered after brushing with toothpastes containing β-TCP followed by bleaching with 10 CP.

Material and Methods
-Experimental Design Sample calculation was calculated with the G*Power program (n=10) with a large effect size (f=0.38)and power of at least ≥80% (β=0.20) for the main effects: group and time, considering the interaction (group × time).The significance level was 5% (α=0.05).The experimental design of the study is presented in Figure 1.
-Staining Protocol Each sample lateral surface was protected with a colorless resistant acid varnish (Risqué Colorless, Taboão da Serra, SP, Brazil) to protect the dentin during the staining protocol, permitting the contact of the staining solution and bleaching gel with the enamel only (25).A solution of black tea (1.8g) (Dr.Oetker LTDA, São Paulo, SP, Brazil) and 100mL of distilled water was dai-e487 ly prepared.The solution was heated for three minutes.Next, samples were infused in boiling water for five minutes.Then, they were submerged in the solution and stored in an oven (at 37 ± 1ºC) for six days, with the solution renewal every 24 hours.Samples were stored in artificial saliva (pH 7.0; composition: Ca 1.5 mmol/L; P 0.9 mmol/L; KCl mmol/L; 0.1mol/L tris buffer) at 37 ± 1ºC for seven days.To achieve color stabilization (7,8,25,26) this solution was replaced daily (7,8,25,26).Finally, prophylaxis was performed on enamel and dentin surfaces using a 2:1 mixture of pumice and water (25).To return the polished and smooth surface of the enamel after the staining protocol, it is preferable a final polishing with #4000 silicon carbide sandpaper (SiC).
-Sample Allocation, randomization, and blinding of the study To assess whether the initial sample was homogeneous and correctly distribute the blocks, based on these data in different groups, the criteria for selecting the samples were based on the mean and standard deviation of the surface microhardness (KHN).Samples with a standard deviation higher (intra-block variability) or lower than 10% of their mean values for individual microhardness were excluded from the study (inter-block variability) (27).Samples were randomized (n=10) into groups according to the bleaching protocol and toothpastes application.The randomization was made after initial color analy-sis, considering the initial values for L* parameter of each sample, which was used to stratify and allocate specimens in all groups, to reduce the initial variability between groups tested (28).Toothpastes was removed from their original packaging and transferred into another vial.A second researcher performed this procedure to ensure blinding of the study, allowing no influence on the knowledge of the treatment being performed in each group.25), was prepared forming a "slurry" solution (1:3) ( 25).An electric toothbrush (Oral-B Professional Care 3000; Oral-B, Schwalbach am Taunus, Germany) (29) was chosen to perform the brushing protocol.To maintain the standardization of toothbrush position, a custom dense silicone (Express™ XT Denso -3M) device was used, allowing the parallelism of the toothbrush and the enamel surface (30).Pressure exerted on the sample surface was normalized at 2.5 N, indicated by a light alert.The toothbrush was triggered for 15 seconds on each surface (31).After this procedure, samples were kept in the "slurry" solution for 2 minutes.Then, they were cautiously washed with distilled water and stored in artificial saliva.The brushing protocol was performed daily for seven days (28,32).Toothbrushing was performed before the bleaching treatment as reported in previous studies (24,32).(Supplement 1) (http://www.medicinaoral.com/medoralfree01/aop/jced_61300_s01.pdf)-Bleaching Protocol Samples were submitted to bleaching treatment with a 10% CP gel (Whiteness Perfect, FGM, Joinville -Santa Catarina/ Brazil) for 14 days, following at-home bleaching technique, with daily application of 4 hours.Subsequently, the gel was removed cautiously with a cotton swab, and the samples were washed abundantly with distilled water to remove remnants of bleaching gel.After each session, the samples were stored in artificial saliva, with daily exchanges.
-Surface Microhardness (KHN) Microhardness analyses were performed with Shimadzu HMV-2000 (Tokyo, Japan).The specimens were positioned with the enamel surface parallel to the Knoop indentation.Then, five indentations were made in the center of each sample, considering a load of 50g, the time of 5 seconds, and a distance of 100µm between each indentation.The arithmetic mean was measured by means of 5 indentations.Enamel indentations can change the topography of the surface, thus, modifying its light reflectance pattern, with negative impact on color analysis.Therefore, KHN was determined 24 hours after bleaching treatment (T3).The bleached groups results were compared with the NC group (32).
-Scanning Electron Microscopy (SEM) Three samples were randomly chosen at the end of the study for qualitative analysis of the enamel.A scanning electron microscope (Jeol, JSM 5600LV, Tokyo, Japan) was used for topographic analysis of the enamel surface at 15 kV and 4000× magnification.Each sample was previously metalized (Bal-Tex SCD 050 sputter coter, Germany) with gold alloy.

-Statistical Analysis
The data were submitted to homogeneity of variance by the Levene test, and normality test using Shapiro-Wilk.Next, descriptive, and exploratory analyses of the data were performed.Next, data were analyzed using generalized linear models for color and surface microhardness with R* statistical software, considering a 5% significance level.*R Core Team (2022).R: A language and environment for statistical computing.R Foundation for Statistical Computing, Vienna, Austria.
• 24 hours after bleaching ∆E* ab and ∆E 00 values were significantly lower in the NC group than in the bleaching groups at time T3 com- pared to times T1 and T2 (p<0.0001).∆E* ab and ∆E 00 did not differ significantly among the five bleaching groups (p > 0.05) (Table 2).
• Whiteness Index for Dentistry (∆WI D ) ∆WI D did not differ significantly between groups at times T1 and T2 (p=0.2637;p=0.5197, respectively).∆WI D in T3 was significantly lower in the NC than in the other groups (p<0.0001).∆WI D did not differ significantly among the five bleaching groups (p>0.05)(Table 3).
-Knoop Microhardness (KHN) Table 4 shows the results of KHN.It can be observed that there was no significant difference in KHN values considering the comparison between NC and the groups bleached with CP 10 (p=0.7585).
-Scanning Electron Microscopy (SEM) β-tricalcium phosphate particles morphology is demonstrated in Figure 3.It can be observed the presence of agglomerated mineralized particles with different sizes ranging from 500 nm to 1500 nm.The enamel in the NC group present polished and smooth appearance (Fig. 4A).The groups that were submitted to bleaching treatment present slight demineralization of the aprismatic enamel layer (Fig. 4B-F).Additionally, the groups treated with toothpastes containing β-TCP shows the presence of whitish spots indicative of mineral deposition in enamel.However, in all images it is observed that the polishing and smoothness of the enamel was properly maintained, similarly to the NC group (Fig. 4A).

Discussion
The first null hypothesis was accepted since there was no significant color alteration after brushing protocol with toothpastes containing β-TCP associated with 10 CP.Extrinsic and intrinsic staining factors determine tooth color (6,21).Extrinsic staining corresponds to pigmentation depositions on the surface of the acquired pellicle from food and habits, such as smoking (21).These pigments are easily removed by brushing with toothpaste.While brushing contributes to a more aesthetic and  less yellowish appearance (6,22), interference with the intrinsic tooth color factors is necessary to achieve a significant color alteration (7).Intrinsic staining is caused by chromogen molecules inside the dentin that absorb light and reflect the color of the tooth surface, due to enamel's translucency (22).Therefore, to promote an effective color alteration, it is necessary to perform bleaching procedures, with CP or HP, in high (in-office bleaching) or low (supervised at-home bleaching) concentrations (3).In this study, groups that underwent bleaching with 10 CP showed color changes when compared to the NC group.Due to its low molecular weight, hydrogen peroxide penetrates the tooth's mineralized structures (4) and breaks down the chromogenic molecules in the dentin through a redox reaction (5).Breaking down and removing these molecules from tooth structure decreases light absorption, resulting in whiter teeth (7).ΔE* ab and ΔE 00 differed significantly in all groups when compared to NC group.Additionally, all groups treated with 10 CP presented statistical similarities.It is possible to conclude that the 14-day, with daily four-hour applications using 10% CP in association with prior brushing with toothpaste containing β-TCP, did not significantly affected the efficacy of the bleaching treatment.
Regarding to the whiteness index (∆WI D ), it was observed that the groups submitted to 10 CP treatment only differed from the NC group in T3 (24 hours after bleaching), which was previously expected, because the exposure only to the toothpaste is not able to cause color alteration, since the toothpastes used in this study do not present bleaching agents in the formulation such as CP and HP.
The second null hypothesis was accepted since there were no significant differences in KHN after brushing followed by bleaching with 10 CP.Tooth bleaching is considered a safe and effective technique when correctly performed (20).However, some studies have reported changes to the enamel surface, such as alterations in enamel´s microhardness and topography due to mineral content loss (8,9).Nevertheless, this study did not identify significant changes in KHN after a bleaching protocol with 10 CP.Lopes et al. 2022 (11) found significant changes in enamel microhardness when brushing was performed with the same toothpastes evaluated in this study.Though, in this previous study the bleaching protocol evaluated used a higher concentration (Whiteness HP 35% FGM, SP, Brazil).This study aimed to compare whether similar results would be found when using lower concentration bleaching gel (10 CP).This study show that 10 CP does not significantly alter the enamel microhardness, in agreement with previous studies that showed no significant changes in the dental enamel surface microhardness and roughness (2).Some factors may have contributed to the maintenance e492 of enamel microhardness.First, the samples were kept in artificial saliva solution throughout the 14 days of experiment, with daily replacement, simulating an oral clinical situation.The artificial saliva solution used in this study ( 32) is a remineralizing solution, which favors mineral deposition on the enamel surface.Secondly, all toothpastes analyzed in this study contained fluoride in its composition.Fluoride is a remineralizing agent commonly used in commercial toothpastes (2,12).In agreement with Juntavee et al. 2021 (12), which emphasizes the capability of mineral recovery provided by fluoride ions present in human saliva, and the use of fluoridated solutions.Finally, the lower bleaching agent concentration (10%) should be considered since a previous study with a higher bleaching agent concentration (35%) reported higher microhardness values (11).
In addition to compounds containing fluoride as remineralizing agents (32), particles such as β-TCP ( 17) have recently been introduced into toothpastes formulations, with the objective to minimize the deleterious effects promoted by bleaching agents on enamel (32).β-TCP composition is based on calcium and phosphate ions that act as a source of mineral components (19).Their mechanism of action occurs when in contact with the teeth moistened surface, breaking the protective barrier, and releasing calcium, phosphate, and fluoride ions, which become available in the oral environment for deposition on the dental enamel surface (12).Therefore, the maintenance of enamel microhardness may reflect the combined effects of the artificial saliva remineralizing effects and the use of toothpastes containing β-TCP, fluoride and a low-concentration bleaching gel (10 CP).Juntavee et al. 2021 (12) discussed that remineralization occurs as a dynamic process, to maintain a balance in the teeth hard tissues.The third null hypothesis was accepted since there were no significant alterations in the dental enamel surface topography regarding polishing and smoothness.Slight mineral deposition was detected on the surface of samples brushed with toothpastes containing β-TCP, represented by whitish spots on the enamel surface.This mineral deposition can be explained by the presence of remineralizing particles on toothpaste formulation (β-TCP and fluoride).However, further studies should be conducted to confirm the presence of this particle on the sample's surfaces.
Recently, de Amoêdo Campos Velo et al. 2020 (16), showed β-TCP on the toothpastes can be interesting to repair active dentin caries lesions.According to the authors, in a long-term process, the continuous using of such toothpastes can be an interesting option to patients at high-risk, such as the elderly population or patients under local/systemic alterations resulting in low salivary flow or compromised quality of saliva, especially when associated with fluoride in the composition, as well as the toothpastes analyzed in the present study.
In addition, as previously discussed, saliva is fundamentally important in the process of dental remineralization.Daily replacement of this solution may have contributed to maintaining the teeth mineral content.Nevertheless, to verify these results in more reliable conditions, further studies are indicated, to evaluate the inorganic content of dental enamel.

Fig. 3 :Fig. 4 :
Fig. 3: Low (1000x) (A) and (B) high magnification (2000x) SEM view of the β-tricalcium phosphate particles present on commercial toothpaste Elmex Sensitive (ES).β-tricalcium phosphate particles are characterized by the presence of agglomerated mineralized particles with different morphology of various irregular sized polygonal crystals that ranges from 500 nm to 1500 nm.

Table 3 :
Mean (standard deviation) of ΔWID, according to the groups, in the times T 1 , T 2 and T 3 .

Table 4 :
Mean (standard derivation) of microhardness (KHN) according to the group.